Summary of work: DNA repair enzymes are activated in response to DNA damage. Alterations in DNA repair enzyme function and thus the rate and extent of repair contribute to malignant transformation. Transcription of the gene encoding DNA polymerizing enzyme beta- polymerase, a repair enzyme, is shown to be inhibited by the human T-cell leukemia virus type I (HTLV-I) trans-activator protein Tax. The resultant inhibition of DNA repair may lead to unrepaired chromosomal damage. A major characteristic of human immunodeficiency virus I (HIVI) infection is the associated immunodeficiency. Recent studies indicated that HIVI gene expression can be enhanced by certain heterologous viral and chemical agents. Mechanisms that induce DNA damage in host cells appear to activate HIV gene expression. Upon irradiation with UV, HIV infected human T-cells detected viral growth 4-5 days earlier than the uninfected cells. Hela cell lines expressing chloramphenicol acetyl transferase (CAT) gene under the control of HIV long terminal repeats (LTRs) showed higher CAT activity upon irradiation with UV. This activation is comparable to that with mitomycin-C or tat HIV trans-activator. It is possible that DNA repair factors, induced or activated by a variety of agents are responsible for triggering a cascade of events which include initial expression of tat protein followed by highly elevated HIV production. DNA double-strand breaks (dsb) are introduced by X-rays and by oxidative metabolism. They confer the highest potential for genetic instability and cell death. So their repair is essential to cell survival. Several radiosensitive mutant cell lines are defective both in DNA dsb repair and in V(D)J recombination. V(D)J recombination is required for the assembly of antigen receptor gene segments and requires both lymphocyte-specific genes and general DNA repair activities. The essential connection between dsb repair and V(D)J recombination was first revealed by analyses of the murine severe combined immunodeficiency mutation (SCID) which are sensitive to ionizing radiation in all cell types because of their deficiency in DNA dsb repair. In SCID lymphocyte precursors, disrupted V(D)J recombination results in T- and B-cell maturational arrest and profound immunodeficiency. Nonlymphoid cells in SCID mice are also hypersensitive to ionizing radiation and other agents which cause dsb. Thus the scid gene encodes or regulates a dsb repair activity expressed in all cell lineages that also plays an essential role in V(D)J recombination. The DNA targeting subunit of DNA-activated protein kinase (DNA-PK) , Ku, may function in both DNA dsb repair and V(D)J recombination. Recent studies have identified products of XRCC7 and XRCC5 dsb repair genes as subunits of the DNA-PK complex. The XRCC 7 gene appears to encode the catalytic subunit of DNA-PK (DNA-PKcs), a 460- KDa serine/threonine protein kinase which belongs to the phosphatidylinositol (PI)-3 kinase family. A deficiency in DNA-PK activity in SCID cells could result in a failure to elicit the appropriate cellular response to DNA damage. Although the DNA-PK subunit p460 has been clearly implicated as the SCID gene, the nature of the mutation that disrupts its function in dsb repair and V(D)J recombination is unknown. SCID mutation is leaky and can result from the abnormal coordination or regulation of dsb repair processes required to complete coding joint formation during lymphoid development, rather than an absent activity. Little is known about the genes and gene products involved in the DNA dsb repair process. Therefore, this project is aimed at further understanding of the regulation of the dsb repair and its correlation with the immunodeficiency. This may provide important insight into the mechanism of action of HIV and/or provide therapeutic strategies. SCID mouse is a model system to study the interaction between DNA repair and immunodeficiency. Gene specific repair and in vitro dsb repair in SCID DNA will be studied with more concentration on the regulation of the dsb repair. Results so far data indicate that the SCID mouse fibroblasts show less gene specific repair than normal fibroblasts. Both SCID and normal cells displayed similar sensitivity to UV irradiation, although SCID mouse had less ability to form colonies (12-42%) compared to normal mouse. The unscheduled DNA synthesis was determined as the 3H/14C-thymidine incorporation of cells exposed to UV compared to that of unexposed cells. Preliminary data indicated no or little repair in both normal or SCID lines in the overall genome. Gene specific repair appears to be deficient in SCID cells compared with normal murine cells. This suggest a possible deficiency in transcription coupled repair in SCID. This is under intense investigation.